Homogeneous Aqueous Solution of Injectable Chitosan

Abstract

The present invention relates to a homogeneous aqueous solution of injectable chitosan containing a chitosan having a degree of acetylation lower than 20%, said solution containing between 0.1 and 3.5% by weight of chitosan, said solution presenting a pH lower than 6.2, and said aqueous solution being capable of forming crystalline particles of chitosan after injection. The present invention also relates to compounds containing such a homogeneous aqueous solution of chitosan. The invention also relates to such compounds for their use as dermatological or cosmetic compounds, or for their use as a medical device, advantageously as a bioresorbable implant.

Claims

1-15. (canceled)

16. A method of filling tissues comprising injecting into said tissues an injectable homogeneous aqueous solution consisting essentially of water, an acid, a chitosan polymer having a degree of acetylation lower than 20%, optionally a compound to readjust pH, optionally an excipient to adjust osmolarity, and optionally a therapeutic compound, wherein said solution containing between 0.1 and 3.5% by weight of the chitosan polymer, said chitosan polymer having a mean molecular weight between 100,000 and 1,000,000 g/mol, said solution having a pH lower than 6.2, and said aqueous solution being capable of forming crystalline particles of chitosan after injection.

17. The method according to claim 16, wherein the solution can be prepared by the following steps: dissolution of the chitosan polymer in water by the addition of an acid, and, readjustment of the pH in order to obtain an aqueous solution having a pH between 5 and 6.2.

18. The method according to claim 17, wherein, during the dissolution step, the acid is added in an amount to dissolve the chitosan polymer.

19. The method according to claim 16, wherein the therapeutic compound is an analgesic compound, a local anesthetic selected from the group consisting of lidocaine, mepivacaine, bupivacaine, and ropivacaine, an angiogenic compound, a vaccine, or an active compound of a growth factor.

20. The method according to claim 19, wherein the composition is formulated to be administered by intradermal or subcutaneous injection.

21. The method according to claim 19, for a dermatological or cosmetic treatment, or a medical treatment, to form a bioresorbable implant.

22. The method according to claim 21, for the repair or reconstruction of tissues of the skin of the face or body.

23. The method according to claim 21, for filling of facial cavities, for creating or increasing the volume of the human face or body, or for the cicatrization of the skin.

24. The method according to claim 21, for filling tissues in surgery, in cosmetic medicine or surgery, in urology, rheumatology, ophthalmology, odontology, or in angiology.

25. The method according to claim 21, to vectorize active ingredients.

26. The method according to claim 16, wherein the chitosan polymer has a mean molecular weight between 250,000 and 1,000,000 g/mol.

27. The method according to claim 17, wherein the acid is a weak acid selected from the group consisting of acetic acid, glycolic acid, lactic acid, glutamic acid, and mixtures thereof.

28. The method according to claim 23, wherein the facial cavities are lines or wrinkles.

29. The method according to claim 25 to vectorize active ingredients through a carrier of vaccines or hormones.

Description

[0099] A crystallinity peak representative of the line (200) of hydrated chitosan (see for example Osorio-Madrazo et al., Biomacromolecules 2010, 11, 1376-1386) around 1.40 .sup.1 is observed after 24 hours in PBS for a solution according to the present invention of low DA (2%, 15% and 15%), at a concentration of 3% by weight (see FIGS. 1a, 1b and 1c).

[0100] On the other hand, for the highest DAs (40% and 55%), which were tested on a purely comparative basis with the invention, no crystallinity is observed at 24 hours nor even at 72 hours; at longer times because the samples are solubilized in PBS and do not crystallize (see FIGS. 2a and 2b),

[0101] The solutions of low DA according to the invention, due to their greater ability to be crystallized, can thus be distinguished by a specific diffraction behavior, in spite of the very low polymer content of the solutions which are composed of more than 97% water.

[0102] After some time in PBS, the solutions of low DA thus become semicrystalline systems. The solutions of low DA according to the invention thus will have a longer filling effect due to the presence of this crystallinity, while the products of high DA will tend to be solubilized and degraded more quickly in tissues.

[0103] Solutions composed of chitosan of mixed DAs as described in the patent applications of the prior art WO 2008/072230 and WO 2009/150651 (see FIGS. 3a and 3b which show the results concerning mixed DA systems of 15% and 40%, and 15% and 55%, respectively) give results similar to solutions containing only chitosan of DA of 40% or 55% (see FIGS. 2a and 2b). These solutions do not crystallize under physiological conditions and are completely solubilized in PBS after 4 days.

EXAMPLE 2: In Vivo study: Evaluation of Performance and Local Tolerance of Injectable Chitosan Solutions Implanted Intradermally in Rabbits

[0104] The objective of the present study is the evaluation of the macroscopic local tolerance (by the evaluation of erythema, edema, necrosis and ulceration) and the performance (according to hardness and diameter criteria) of 6 test formulations, in comparison with 3 reference products, after intradermal implantation in rabbits.

Elements Tested

[0105] The following compositions were tested: aqueous chitosan solutions, having a concentration of 3% by weight in mixture with 9% NaCl+0.3% lidocaine, sterilized by autoclave at 121 C. for 15 minutes.

[0106] The various DAs are Obtained by reacetylation of a squid chitosan (Mahtani Chitosan Veraval, India) of DA 3.5%, Mw approximately 400,000 g/mol, purified by filtration of a chitosan acetate solution at a concentration of 0.5% by weight of polymer through a 0.45 m filter. The solution is then lyophilized.

[0107] In a reactor, and with mechanical stirring (50 rpm), the chitosan lyophilizate is dissolved in deionized water using the stoichiometric amount of acetic acid necessary to the protonation of NH.sub.2 sites. Various concentrations (0.5 w % to 3 w %) were studied.

[0108] The pH of each solution was controlled, and is in all cases between 5 and 5.5 (as a function of the chitosan concentration).

[0109] Test 1: chitosan C=3 w %, DA=5%

[0110] Test 2: chitosan C=3 w %, DA=15%

[0111] Test 3: chitosan C=3 w %, DA=40%

[0112] Test 4: chitosan C=3 w %, DA=55%

[0113] Test 5: chitosan C=3 w %, DA=5%+40%

[0114] Test 6: chitosan C=3 w %, DA=15%+55%

Reference Elements

[0115] Ref. 1: Restylane Perlane Lidocaine (29G needle), identified as: ref. 1Restylane
Ref. 2: Ultra Juvederm 4 (27G needle), identified as: ref 2Juvederm
Ref. 3: New Fill/Sculptra (26G needle), identified as: ref 3NewFill.

Test System

[0116] Species: rabbit
Strain: New Zealand white

Source: Charles River Laboratories

[0117] Health status: IOPS (SPF)
Number of animals: 8+1 reserve
Sex: female
Age on arrival: 18 weeks

Implantations

[0118] Preimplant procedures:

[0119] At Day 0, the animals were weighed, examined and then anaesthetized, according to the following protocol: [0120] Ketamine (Ketamine 1000-VIRBAC) 30 mg/kg (0.3 ml/kg) [0121] +Medetomidine (Domitor-Janssen Animal Health) 0.1 mg/kg (0.1 ml/kg) [0122] Intramuscular (IM) injection in one thigh. [0123] The dorsal zone was cropped with care (cropped again as needed thereafter for observations.)

[0124] Implantation procedure:

[0125] Six injections were given per animal, on the dorsal zone. Care was taken not to inject too closely to the area of the nape of the neck and shoulders, so that the handling of the animal did not damage the sites.

[0126] Each site was marked by a tattoo, and then injected with 200 l of product.

Follow-up of Animals after Implantation

[0127] Daily observations

[0128] Observations were made each day by the personnel responsible for daily care (feeding, watering, cleaning, etc.),

[0129] They included a weighing of the animal, a complete physical examination, and a quick behavioral observation during handling.

[0130] Thorough clinical exams

[0131] Thorough clinical exams were carried out by the veterinarian, the Study Director or his deputy, generally when a consequent anomaly was noted during a daily observation or a basic clinical exam.

[0132] They included weighing, as well as the measuring of respiratory and cardiac rates, and the taking of rectal temperature.

[0133] The lymphatic, circulatory, respiratory, digestive, musculoskeletal and nervous systems, as well as the skin and the raucous membranes, were examined.

[0134] Macroscopic observations

[0135] The observations took place at the following times:

[0136] Day 0 (post-implantation), T+24 hours, T+48 hours, Day 4. [0137] The animal was photographed from above, while ensuring that the tattoo and. all of the sites were visible. [0138] The implantation sites were evaluated visually or manually by means of a scoring grid. [0139] The diameter of the sites was measured using a caliper.

[0140] The parameters evaluated were: the formation of edema. and erythema, the phenomena of ulceration and necrosis localized at the implantation sites, as well as the hardness and diameter of the sites.

[0141] Scale for observations of edema/erythema/ulcer/necrosis:

[0142] (0) absent /(1) mild/(2) moderate/(3) marked/(4) severe

Euthanasia and Sampling

[0143] At Day 2, five of the eight animals were anaesthetized and then given an intracardiac injection of sodium pentobarbital (DolethalVETOQUINOL).

[0144] The implantation sites were removed and placed in labeled histology cassettes.

[0145] The samples from rabbits 1 to 3 were preserved in formol before treatment for histological study.

[0146] The samples from rabbits 4 and 5 were preserved in pure sterile water. A few hours later, the implant was extracted from the tissues for analysis under a synchrotron beam (WARS technique, ESRF Grenoble, D2AM beamline). An explant fragment was placed in a capillary tube filled with water and then observed by synchrotron x-ray diffraction, using a 16 keV (=0.7749 ) monochromatic beam.

[0147] At Day 4, the three remaining animals were anaesthetized and then given an intracardiac injection of sodium pentobarbital (DolethalVETOQUINOL).

[0148] The implantation sites were removed and placed in labeled histology cassettes. These samples were preserved in formol before treatment for histological study.

Results

[0149] Clinical observations:

[0150] In all cases, during the first 4 days, edema and erythema increase as DA increases. The solutions with DA of 5% and 15% in certain animals induce edema and erythema scores of 0, while the solutions with DA40% result in edema and erythema scores of 3 or 4. The solutions with DA of 5% and 15% according to the present invention are the only ones that did not induce systematic necrosis at the implant sites.

[0151] It is important to note that the implant composed of chitosan with a DA of 55% is no longer palpable after 4 days, and that after 2 days it was not possible to remove it from the tissues for analysis by X-ray diffraction.

[0152] The example of two rabbits (R4 and R5) having received the test formulations is presented in FIGS. 4aand 4b.

[0153] FIGS. 4a and 4b show photographs of the injection sites of rabbits 4 and 5, respectively, 24 hours after implantation:

[0154] A: Test 1: chitosan C=3 w %, DA=5%

[0155] B: Test 2: chitosan C=3 w %, DA=15%

[0156] C: Test 3: chitosan C=3 w %, DA=40%

[0157] D: Test 4: chitosan C=3 w %, DA=55%

[0158] E: Test 5: chitosan C=3 w %, DA=5%+40%

[0159] F: Test 6: chitosan C=3 w %, DA=15%+55%

[0160] The solutions prepared according to the present invention induce a limited inflammatory response in comparison with the solutions containing chitosans of high DA or mixtures of chitosans containing in particular a high DA chitosan as described in patent applications WO 2008/072230 and WO 2009/150651. Moreover, as suggested by the complete disappearance of the implant with a degree of acetylation equal to 55% after only 4 days, the use of a high DA chitosan does not confer a satisfactory bioresorption time for the applications concerned.

Study of Explants by Synchrotron Beam

[0161] FIGS. 5 to 7 represent the results of the study by synchrotron x-ray diffraction beam of chitosan explants 24 hours after intradermal implantation of the solutions.

[0162] FIGS. 5a and 5b represent the results of a chitosan solution according to the invention (site A, test 1), with a low DA equal to 5%, after implantation of the solution in Rabbit 4 and Rabbit 5, respectively.

[0163] FIGS. 5c and 5d represent the results of a chitosan solution according to the invention (site B, test 2), with a low DA equal to 15%, after implantation of the solution in Rabbit 4 and Rabbit 5, respectively.

[0164] FIGS. 6a and 6b represent the results of a comparative chitosan solution (site C, test 3), with a high DA equal to 40%, after implantation of the solution in Rabbit 4 and Rabbit 5, respectively.

[0165] FIGS. 7a and 7b represent the results of a comparative chitosan solution (site E, test 5), with a mixture of low DA and high DA: 5%+40%, after implantation of the solution in Rabbit 4 and Rabbit 5, respectively.

[0166] FIGS. 7c and 7d represent the results of a comparative chitosan solution (site F, test 6), with a mixture of low DA and high DA: 15%+55%, after implantation of the solution in Rabbit 4 and Rabbit 5, respectively.

[0167] The intensity diffracted by the explants is given as a function of the scattering vector q=(4sin )/, where 2 is the angle of diffraction (between the incident beam and the diffracted beam) and after subtraction of the intensity diffracted by the capillary tube filled with water alone, so as to best subtract the contribution of the water and the container to the diffraction.

[0168] In addition to the amorphous halo residue due to water, a well-defined crystallinity peak representative of the 200 line of hydrated chitosan around 1.40 .sup.1 for the explants of low DA (5% and 15%) is observed, whereas in the other cases it is weakly perceptible (DA 40%, DA mixture 5%+40%) or completely absent (DA 15%+55%). The low DA solutions, due to their greater ability to be crystallized, can thus be distinguished by a specific diffraction behavior, in spite of the very low polymer content of the solutions which are composed of more than 97% water.

[0169] After injection in the dermis, these low DA solutions thus become semicrystalline systems. A longer filling effect can thus be expected by virtue of the presence of this crystallinity, whereas the high DA products will tend to be solubilized and degraded more quickly in tissues.

[0170] The solutions composed of chitosan of mixed DAs as described in the patent applications of the prior art WO 2008/072230 and WO 2009/150651 give results similar to the solutions containing only chitosan of DA of 40% or 55%: these solutions do not crystallize, or crystallize very little, in tissues and, for the DA of 55%, they are no longer observable macroscopically within four days.

[0171] The crystallinity developed in situ indeed makes it possible to extend the bioresorption time, and is thus of great advantage for the applications concerned.

EXAMPLE 3: In Vivo study Evaluation of the Performance and Local Tolerance of Injectable Chitosan Solutions, Implanted Subcutaneously in Rats

[0172] The objective of the present study is the evaluation of macroscopic local tolerance (by the evaluation of erythema, edema, necrosis and ulceration) and of performance (according to hardness criteria) of 2 test formulations, in comparison with 1 reference product, after subcutaneous injection in rats.

Elements Tested

[0173] The following compositions were tested: aqueous chitosan solutions, having a concentration of 3% by weight in mixture with 9%.sub.0 NaCl+0.3% lidocaine, sterilized by autoclave at 121 C. for 15 minutes.

[0174] The chitosan used is a squid chitosan (Mahtani Chitosan Veraval, India) of DA 2%, Mw (molar weight) approximately 400,000 g/mol, purified by filtration of a chitosan acetate solution at a concentration of 0.5% by weight of polymer through a 0.45 m filter. The solution is then lyophilized.

[0175] In a reactor, and with mechanical stirring (50 rpm), the chitosan lyophilizate is dissolved in water for injection using the stoichiometric amount of acetic acid necessary to the protonation of NI-I.sub.2 sites. The concentration 3 w % was studied.

[0176] The pH of each solution was controlled, and is in all cases between 5 and 6.2.

[0177] Test 1: chitosan C=3 w %, DA=2%, pH=5.

[0178] Test 2: chitosan C=3 w %, DA=2%, pH=6.

Reference Elements

[0179] Ref 1: Restylane Perlane Lidocaine (29G needle)

Test System

Species: rat

Strain: Sprague Dawley

[0180] Number of animals: 6
Sex: male
Age on arrival: between 7 and 8 weeks
Weight on arrival: between 200 and 220 grams

Implantations

[0181] Preimplant procedures:

[0182] At Day 0, the animals were weighed, examined and then anaesthetized, according to the following protocol:

Intraperitoneal injection (1 ml/100 g of weight of the animal) of a sodium pentobarbital dilution (CEVA ANIMAL HEALTH100 ml at 54.7 mg/ml) in a ratio of 6 ml for 44 ml of physiological saline.
The dorsal zone was cropped with care (cropped again as needed).
No antibiotic treatment was. given.

[0183] Implantation procedure:

[0184] Four subcutaneous injections using sterile glass syringes with sterile needles were given per animal, on the dorsal zone.

[0185] Each implantation site was marked by a tattoo, and then injected with 100 l of product.

[0186] The injection sites were randomized with the criterion that each animal received at least one injection per formulation (test 1, test 2 and Ref. 1).

Follow-Up of Animals after Implantation

[0187] Daily observations

[0188] Observations were made each day by the personnel responsible for daily care (feeding, watering, cleaning, etc.).

[0189] They included a weighing of the animal, a complete physical examination, and a quick behavioral observation during handling.

[0190] Macroscopic observations

[0191] The observations took place at the following times:

[0192] Day 0 (post-implantation), Day 2 (T+48 hours), Day 4 (T+96 hours).

[0193] The implantation sites were evaluated visually or manually by means of a scoring grid.

[0194] The parameters evaluated were: the formation of edema and erythema, the phenomena of ulceration and necrosis localized at the implantation sites, as well as hardness.

[0195] Scale for observations of edema./erythema/ulcer/necrosis: (0) absent/(1) mild/(2) moderate/(3) marked/(4) severe

Euthanasia and Sampling

[0196] At Day 4, the animals were anaesthetized and then given a sodium pentobarbital injection (2 ml undiluted, IP). The implantation sites were removed so as to include the lesion and a contiguous uninjured zone, each sample comprising all the layers of the skin to the muscle. The samples were fixed in 4% aqueous formaldehyde solution for 48 hours.

Results

[0197] Clinical observations:

[0198] All the animals appeared and behaved normally during the entire observation period and their weight remained stable.

[0199] The visual and manual evaluation of the implant sites did not reveal any difference related to the formulations tested.

[0200] No erythemas induced by the solutions according to the present invention injected subcutaneously were observed.

[0201] The volumes observed are not related to an irritating effect of the solutions tested but are mechanical in origin (implant not reabsorbed).

[0202] In the experimental conditions adopted, the 2 solution formulations according to the present invention were well tolerated locally.

[0203] The evaluation of the macroscopic local tolerance and performance of test formulations 1 and 2 was comparable to that of the reference formulation Ref 1.

[0204] No significant adverse reaction such as significant necrosis and ulceration of the skin was observed. The solutions prepared according to the present invention thus induce a limited inflammatory response.

[0205] Histological examination of the implants

[0206] The samples were fixed at least 24 hours before being dried.

[0207] One section (thickness 3 to 5 m) was made per block. The slides were stained with hematoxylin-eosin.

[0208] Twenty-four virtual slides were analyzed.

[0209] The histological appearance of the implants is very different between the reference implant (Restylane/Perlane/Lidocaine) and the test implants (Test 1 and Test 2). Whereas the reference implant was homogeneous, the test implants had an appearance that was either micro-globular (variable diameter, generally between 5 and 15 m -'Test 1), or of the coagulum type (Test 2).

[0210] The reaction of the host to formulations Test 1 and Test 2 was generally limited to the hypodermis under the platysma muscle, consistent with fibroplasia/granulation tissue and mild-to-moderate inflammation surrounding the implant. This reaction consisted of granulation tissue rich in collagen fibers in the process of maturation and infiltration by mononucleated cells mostly consisting of monocytes/histiocytes and lymphocytes, with occasionally plasmocytes, but mostly without granulocytes.

[0211] Based on these criteria, a classification of weak reaction of the host (score 1) was observed for the reference item, an intermediate reaction (score 2) for formulations Test 1 and Test 2, with however a more marked host reaction in the case of item Test 1.

[0212] The solutions composed of chitosan according to the invention give results similar to the reference solution in terms of tolerance. On the other hand, just as in Example 2, after subcutaneous injection, these low DA solutions become semicrystalline systems. A longer filling effect by virtue of the presence of this crystallinity can thus be expected.